Biology subjects

Kubikova, J.

Publications and source records attributed to Kubikova, J..

3 recordsLinked to original sources

bioRxiv2025

Probing direct interactions between nuclear proteins in cells with nxReLo

Nearly all biological processes depend on protein-protein interactions (PPIs). While various methods exist to study these interactions, investigating those that involve nuclear proteins, including structurally complex proteins containing long disordered regions, remains a significant challenge. Here, we developed nxReLo, a simple and fast cell culture-based colocalization assay, designed to identify and characterize interactions between nuclear proteins. PIWI-interacting RNAs (piRNAs) safeguard germline genome integrity, and the PPIs that facilitate piRNA production are therefore essential for animal reproduction, yet remain incompletely understood. We used nxReLo to investigate interactions between members of the Drosophila melanogaster Rhino-Deadlock-Cutoff (RDC) complex and two associated components, Bootlegger and Moonshiner, a nuclear protein network required for piRNA expression. We demonstrate the utility of the nxReLo assay by systematically screening pairwise interactions within the RDC network and by assembling a multiprotein complex from multiple components. By combining nxReLo assays with AlphaFold structural prediction, we characterized the Cutoff-Deadlock and the Bootlegger-Deadlock complexes in detail, providing molecular and structural insights. Specifically, we refined the domains involved in the interaction and identified interface point mutations that interfered with complex formation, validating the predicted structures. In conclusion, nxReLo facilitates rapid and simple testing of direct interactions between nuclear proteins in a cellular context, which is particularly important when working with structurally challenging proteins or when established interaction assays prove unsuccessful.

biochemistry↗

bioRxiv2023

Structural basis for binding of Smaug to the GPCR Smoothened and to the germline inducer Oskar

Drosophila Smaug and its orthologs comprise a family of mRNA repressor proteins that exhibit various functions during animal development. Smaug proteins contain a characteristic RNA-binding sterile- motif (SAM) domain and a conserved but uncharacterized N-terminal domain (NTD). Here, we resolved the crystal structure of the NTD of the human SAM domain-containing protein 4A (SAMD4A, a.k.a. Smaug1) to 2.0 [A] resolution, which revealed its composition of a homodimerization D-subdomain and a subdomain with similarity to a PHAT domain. Furthermore, we show that Drosophila Smaug directly interacts with the Drosophila germline inducer Oskar and with the Hedgehog signaling transducer Smoothened through its D-PHAT domain. We determined the crystal structure of the D-PHAT domain of Smaug in complex with a Smoothened -helical peptide to 1.61 [A] resolution. The peptide binds within a groove that is formed by both the D- and PHAT subdomains. Structural modeling supported by experimental data suggested that an -helix within the disordered region of Oskar binds to the D-PHAT domain in a mode similar to Smoothened. Together, our data uncover the N-terminal D-PHAT domain of Smaug as peptide-binding domain.

biochemistry↗

bioRxiv2022

RNA binding proteins Smaug and Cup induce CCR4-NOT-dependent deadenylation of the nanos mRNA in a reconstituted system

Posttranscriptional regulation of the maternal nanos mRNA is essential for the development of the anterior - posterior axis of the Drosophila embryo. The nanos RNA is regulated by the protein Smaug. Binding to Smaug recognition elements (SREs) in the nanos 3-UTR, Smaug nucleates the assembly of a larger repressor complex including the eIF4E-T paralog Cup and five additional proteins. The Smaug-dependent complex represses translation of nanos and induces its deadenylation by the CCR4-NOT deadenylase. Here we report an in vitro reconstitution of the Drosophila CCR4-NOT complex and Smaug-dependent deadenylation. We find that Smaug by itself is sufficient to cause deadenylation by the Drosophila or human CCR4-NOT complexes in an SRE-dependent manner. CCR4-NOT subunits NOT10 and NOT11 are dispensable, but the NOT module, consisting of NOT2, NOT3 and the C-terminal part of NOT1, is required. Smaug interacts with the C-terminal domain of NOT3. Both catalytic subunits of CCR4-NOT contribute to Smaug-dependent deadenylation. Whereas the CCR4-NOT complex itself acts distributively, Smaug induces a processive behavior. The cytoplasmic poly(A) binding protein (PABPC) has but a minor effect on Smaug-dependent deadenylation. Among the additional constituents of the Smaug-dependent repressor complex, Cup also facilitates CCR4-NOT-dependent deadenylation, both independently and in cooperation with Smaug.

biochemistry↗